U.S. patent number 10,228,370 [Application Number 15/587,907] was granted by the patent office on 2019-03-12 for recombinant trypanosoma cruzi jl7 antigen variants and their use for detecting chagas disease.
This patent grant is currently assigned to Roche Diagnostics Operations, Inc.. The grantee listed for this patent is Roche Diagnostics Operations, Inc.. Invention is credited to Dieter Roessler, Barbara Upmeier, Toralf Zarnt.
United States Patent |
10,228,370 |
Roessler , et al. |
March 12, 2019 |
Recombinant Trypanosoma cruzi JL7 antigen variants and their use
for detecting Chagas disease
Abstract
The invention concerns variants of JL7 antigens that are
suitable for detecting antibodies against Trypanosoma cruzi
(causing Chagas disease) in an isolated biological sample. These
antigens comprise a JL7 specific amino acid sequence, said JL7
specific sequence consisting of two copies of SEQ ID NO. 2, wherein
each of said two copies has an amino acid identity of at least 90%
to SEQ ID NO.2 and wherein no further Trypanosoma cruzi specific
amino acid sequences are present in said polypeptide. The invention
also concerns a composition of polypeptides useful for the
detection of antibodies against Trypanosoma cruzi that comprises
the above characterized JL7 antigen along with at least one of T.
cruzi polypeptides 1F8, Cruzipain, KMP-11 and PAR-2. Moreover, it
relates to a method for producing JL7 antigen as well as to
diagnostic methods for detecting T. cruzi antibodies using the JL7
polypeptide. In addition, the invention concerns a reagent kit
comprising said JL7 polypeptides or composition of Trypanosoma
cruzi polypeptides.
Inventors: |
Roessler; Dieter (Kirchseeon,
DE), Upmeier; Barbara (Iffeldorf, DE),
Zarnt; Toralf (Penzberg, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc. |
Indianapolis |
IN |
US |
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Assignee: |
Roche Diagnostics Operations,
Inc. (Indianapolis, IN)
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Family
ID: |
54478742 |
Appl.
No.: |
15/587,907 |
Filed: |
May 5, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170248598 A1 |
Aug 31, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/EP2015/075692 |
Nov 4, 2015 |
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Foreign Application Priority Data
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Nov 6, 2014 [EP] |
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14192004 |
Mar 27, 2015 [EP] |
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15161274 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/44 (20130101); G01N 33/564 (20130101); G01N
33/56905 (20130101); C07K 2319/21 (20130101); G01N
2469/20 (20130101); C07K 2319/40 (20130101); G01N
2333/44 (20130101); C07K 2319/70 (20130101); G01N
2800/26 (20130101) |
Current International
Class: |
G01N
33/569 (20060101); C07K 14/44 (20060101); G01N
33/564 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0976763 |
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Nov 2003 |
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EP |
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2009/017736 |
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Feb 2009 |
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WO |
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2010/142829 |
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Dec 2010 |
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WO |
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Other References
Bottino, Carolina G. et al., Chagas disease-specific antigens:
characterization of epitopes in CRA/FRA by synthetic peptide
mapping and evaluation by ELISA-peptide assay, BMC Infectious
Diseases, 2013, 10 pps., vol. 13, No. 568. cited by applicant .
Camussone, Cecilia et al., Comparison of Recombinant Trypanosoma
cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as
Sensitizing Antigens for Immunodiagnosis, Clinical and Vaccine
Immunology, 2009, pp. 899-905, vol. 16, No. 6. cited by applicant
.
Chiaramonte, M. G. et al., Polymerase chain reaction reveals
Trypanosoma cruzi infection suspected by serology in cutaneous and
mucocutaneous leishmaniasis patients, Acta Tropica, 1999, pp.
295-308, vol. 72. cited by applicant .
Cotrim, Paulo C. et al., Organization and expression of the gene
encoding an immunodominant repetitive antigen associated to the
cytoskeleton of Trypanosoma cruzi, Molecular and Biochemical
Parasitology, 1995, pp. 89-98, vol. 71. cited by applicant .
Da Silveira, Jose Franco et al., Chagas Disease: recombinant
Trypanosoma cruzi antigens for serological diagnosis, Trends in
Parasitology, 2001, pp. 286-291, vol. 17, No. 6. cited by applicant
.
Fernandez-Villegas, Ana et al., Short-term follow-up of chagasic
patients after benznidazole treatment using multiple serological
markers, BMC Infectious Diseases, 2011, 7 pages, vol. 11, No. 206.
cited by applicant .
International Search Report dated Jan. 14, 2016, in Application No.
PCT/EP2015/675692, 4 pages. cited by applicant .
Longhi, Silvia A. et al., Short Report: Evaluation of In-House
ELISA Using Trypanosoma cruzi Lysate and Recombinant Antigens for
Diagnosis of Chagas Disease and Discrimination of Its Clinical
Forms, American J. Trop. Med. Hyg., 2012, pp. 267-271, vol. 87, No.
2. cited by applicant .
Marcipar, Ivan S. and Lagier, Claudia M., Advances in Serological
Diagnosis of Chagas' Disease by Using Recombinant Proteins, Current
Topics in Tropical Medicine, 2012, pp. 273-298. cited by applicant
.
Thomas, M. C. et al., Mapping of the antigenic determinants of the
T. cruzi kinetoplastid membrane protein-11. Identification of a
linear epitope specifically recognized by human Chagasic sera,
Clinical and Experimental Immunology, 2001, pp. 465-471, vol. 123.
cited by applicant .
Umezawa, Eufrosina S. et al., An improved serodiagnostic test for
Chagas' disease employing a mixture of Trypanosoma cruzi
recombinant antigens, Transfusion, 2003, pp. 91-97, vol. 43. cited
by applicant .
Umezawa, Eufrosina S. et al., Evaluation of Recombinant Antigens
for Serodiagnosis of Chagas' Disease in South and Central America,
Journal of Clinical Microbiology, 1999, pp. 1554-1560, vol. 37, No.
5. cited by applicant .
Valiente-Gabioud, Ariel A. et al., Effect of repetitiveness on the
immunogenicity and antigenicity of Trypanosoma cruzi FRA protein,
Experimental Parasitology, 2011, pp. 672-679, vol. 127, No. 3.
cited by applicant.
|
Primary Examiner: Baskar; Padmavathi
Attorney, Agent or Firm: Roche Diagnostics Operations,
Inc.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of International Application No.
PCT/EP2015/075692 filed Nov. 4, 2015, which claims priority to
European Application Nos. 14192004.1 filed Nov. 6, 2014 and
15161274.4 filed Mar. 27, 2015, the disclosures of which are hereby
incorporated by reference in their entirety.
Claims
The invention claimed is:
1. A polypeptide suitable for detecting antibodies against
Trypanosoma cruzi in an isolated biological sample comprising
Trypanosoma cruzi JL7 specific amino acid sequence consists of SEQ
ID NO. 4, and wherein no further Trypanosoma cruzi specific amino
acid sequences are present in said polypeptide.
2. A composition of polypeptides suitable for detecting antibodies
against Trypanosoma cruzi antigens in an isolated biological sample
comprising a polypeptide according to claim 1 and at least one
Trypanosoma cruzi polypeptide selected from the group consisting of
1F8, Cruzipain, KMP-11 and PAR-2.
3. A reagent kit for the detection of anti-Trypanosoma cruzi
antibodies, comprising a Trypanosoma cruzi JL7 polypeptide
according to claim 1 or of a composition according to claim 2.
Description
BACKGROUND OF THE DISCLOSURE
Trypanosoma cruzi is a flagellate protozoan that causes a tropical
parasitic disease called Chagas disease. In the course of Chagas
disease the infected patient typically passes an acute phase
followed by a chronic phase with varying symptoms ranging between
mild and life-threatening. Only in the initial phase the patient is
responsive to antiparasitic treatment. Some patients develop
cardiac or intestinal complications leading to death.
T. cruzi is commonly transmitted to humans and other mammals by an
insect vector, the blood-sucking "kissing bugs" of the subfamily
Triatominae (family Reduviidae; in German: "Raubwanzen"). The
disease may also be spread through blood transfusion and organ
transplantation, ingestion of food contaminated with parasites, and
from a mother to her fetus. To prevent further spread of Chagas
disease it is important to screen blood donations and medical
products based on blood for the presence of the parasite and for
antibodies against T. cruzi, in particular in mainly infected
territories like Mexico, Central and South America and in the
Southern United States.
Currently, several serologic diagnostic methods are available to
detect infections with T. cruzi, e.g. detection of antibodies
against T. cruzi by indirect immunofluorescence, indirect
hemagglutination, complement fixation, immunoblot techniques and
ELISAs. Also methods of molecular biology (e.g. PCR) and elaborate
xenodiagnostic methods are applied. In xenodiagnostics a
vector-transmitted infection a laboratory-reared, pathogen-free
insect (here: the kissing bug) is allowed to suck blood from a
patient. The intestinal contents of the insect are then examined
for the presence of the pathogen (here: Trypanosoma cruzi).
Each of these methods shows its own weaknesses and strengths with
regard to sensitivity and specificity and accordingly there is no
gold standard method available so far.
In the beginning of Chagas assay development for detection of
antibodies native antigen lysates were applied and are still being
used. However, using lysates only one of the three development
stages of T. cruzi is represented in this antigen composition so
that there is a certain likelihood to miss infections of the two
other stages. More modern assays apply mixtures of recombinant
antigens, representing all stages of T. cruzi infection.
Serological assays for detecting antibodies against Trypanosoma
cruzi antigens have been widely described in prior art literature.
For example Silveira et al. (Trends in Parasitology 2001, Vol. 17
No. 6, p. 286-291) describes T. cruzi recombinant antigens relevant
for serodiagnosis have been isolated by several laboratories.
Several of these genes have tandemly repeated sequences resulting
in proteins showing repeated amino acid sequence motifs.
One of the antigens that is widely used in serological assays for
detection of antibodies against T. cruzi is JL7, UniProt database
entry no. Q4CS87, also described by the synonyms FRA, Agl and H49
(for review see e.g. Cotrim et al. 1995, Mol Biochem Parasitol 71,
89-98 and Umezawa et al. 1999, J Clin Microbiol, 1554-1560). Also
Silveira et al. (supra) describe FRA (JL7) in several mixture of
various additional T. cruzi antigens for use in ELISA assays. No
specific JL7-related polypeptides are disclosed.
Valiente-Gabioud et al. (Exp. Parasitol. 2011, 127, p. 672-679)
discuss the effect of repetitiveness on the immunogenicity and
antigenicity of T. cruzi FRA (JL7) protein. It is recommended to
use four repetitive repeats to get satisfying ELISA signals from
human infected sera. The problem of assay specificity is not
addressed and no specific JL7 polypeptides are disclosed.
All antigen variants based on the above mentioned JL7 sequence
harbor repeats of 68 amino acids that are highly conserved between
strains and isolates of T. cruzi. JL7 is a putative calpain
cysteine peptidase of 226 amino acids comprising about 3.5 repeats
of said motif of 68 amino acids (see FIG. 1). However, antigens
harboring several identical or very similar repeats are often prone
to interferences caused by non-specific binding of IgM or IgG
molecules or rheumatic factors present in a sample to be analyzed
for the presence of anti-T. cruzi antibodies.
The problem therefore can be seen in providing a recombinant JL7
antigen composition and diagnostic method for detecting infections
with Trypanosoma cruzi that overcome the disadvantages with respect
to interference susceptibility.
The problem is solved by the current invention as specified in the
claims. The claimed antigens and compositions as well as diagnostic
methods provide an immunodiagnostic solution with high specificity
and sensitivity and reliable reproducibility.
SUMMARY OF THE INVENTION
The present invention concerns variants of JL7 antigens that are
suitable for detecting antibodies against Trypanosoma cruzi JL7
antigen in an isolated biological sample. These antigens comprise a
JL7 specific amino acid sequence, said JL7 specific sequence
consisting of two copies of SEQ ID NO. 2, wherein each of said two
copies has an amino acid identity of at least 90% to SEQ ID NO. 2
and wherein no further Trypanosoma cruzi specific amino acid
sequences are present in said polypeptide. The invention also
concerns a composition of polypeptides useful for the detection of
antibodies against Trypanosoma cruzi that comprises the above
characterized JL7 antigen along with at least one of T. cruzi
polypeptides 1F8, Cruzipain, KMP-11 and PAR-2. Moreover, the
invention relates to a method for producing JL7 antigen as well as
to diagnostic methods for detecting T. cruzi antibodies using the
JL7 polypeptide. In addition, the invention concerns a reagent kit
comprising said JL7 polypeptides or composition of Trypanosoma
cruzi polypeptides.
Legend to the Disclosed Amino Acid Sequences
SEQ ID NO. 1 shows a partial sequence of T. cruzi protein JL7
(UniProt entry Q4CS87), also known as FRA, Agl, H49; full
descriptive name: calpain cysteine peptidase, putative. SEQ ID NO.
1 shows amino acid positions 62-287 of the above UniProt database
entry, resulting in a polypeptide with a length of 226 amino acids.
The full length protein comprises amino acids 1 to 1275.
TABLE-US-00001 MEQERRQLLE KDPRRNAREI AALEESMNAR AQELAREKKL
ADRAFLDQKP EGVPLRELPL DDDSDFVAME QERRQLLEKD PRRNAKEIAA LEESMNARAQ
ELAREKKLAD RAFLDQKPEG VPLRELPLDD DSDFVSMEQE RRQLLEKDPR RNVQKIADLE
ESMNARAQEL AREKKLADRA FLDQKPEGVS LRELPLDDDS DFVSMEQERR QLLEKDPRKN
VQIVAD
SEQ ID NO. 2 shows antigen JL7short1, also shown as amino acid
positions 1-68 of SEQ ID NO.1
TABLE-US-00002 MEQERRQLLE KDPRRNAKEI AALEESMNAR AQELAREKKL
ADRAFLDQKP EGVPLRELPL DDDSDFVA
SEQ ID NO. 3 shows antigen JL7short2, also shown as amino acid
positions 69-136 of SEQ ID NO.1
TABLE-US-00003 MEQERRQLLE KDPRRNAKEI AALEESMNAR AQELAREKKL
ADRAFLDQKP EGVPLRELPL DDDSDFVS
SEQ ID NO. 4 shows antigen JL7short1+2 which corresponds to a
combination of SEQ ID NOs. 2 and 3, also shown as amino acid
positions 1-136 of SEQ ID NO.1
TABLE-US-00004 MEQERRQLLE KDPRRNAREI AALEESMNAR AQELAREKKL
ADRAFLDQKP EGVPLRELPL DDDSDFVAME QERRQLLEKD PRRNAKEIAA LEESMNARAQ
ELAREKKLAD RAFLDQKPEG VPLRELPLDD DSDFVS
SEQ ID NO. 5 shows antigen JL7short3+4 which corresponds to amino
acid positions 137-226 of SEQ ID NO.1
TABLE-US-00005 MEQERRQLLE KDPRRNVQKI ADLEESMNAR AQELAREKKL
ADRAFLDQKP EGVSLRELPL DDDSDFVSME QERRQLLEKD PRKNVQIVAD
SEQ ID NO. 6 shows antigen JL7short3 which corresponds to amino
acid positions 137-204 of SEQ ID NO.1
TABLE-US-00006 MEQERRQLLE KDPRRNVQKI ADLEESMNAR AQELAREKKL
ADRAFLDQKP EGVSLRELPL DDDSDFVS
SEQ ID NO. 7 shows a hexa-histidine tag that can be added to the
N-terminal or preferably to the C-terminal end the polypeptides
according to the invention. The tag is used to facilitate protein
purification.
TABLE-US-00007 GGGSGGGLEH HHHHH
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Structure of repeats in complete JL7 amino acid sequence.
Upper section: All four repeats are aligned showing amino acid
identity (bold letters) and differences (normal underlined
letters). Lower section: schematic drawing of JL7 variants and
relative position of their repeat domain(s). JL7short1+2 represents
a JL7 antigen according to this invention.
FIG. 2: Far-UV CD spectra of JL7, JL7short1, JL7short2, JL7short1+2
and JL7short3+4. The spectra were recorded on a Jasco-720
spectropolarimeter in a thermostatted cell holder at 20.degree. C.
The protein concentration was 0.2 mg/ml in a 0.1 cm cuvette. The
buffer was 10 mM potassium phosphate pH 7.5. Band width was 1 nm,
resolution was 1 nm, the scanning speed was 50 nm/min at a response
of 2 s. Spectra were recorded 8 times and averaged in order to
improve the signal-to-noise ratio. The signal was converted to
molar ellipticity (given in deg cm.sup.2 dmol.sup.-1). The spectra
of JL7 and JL7short1+2 point to proteins with high content of
.alpha.-helical structural elements (signal bands at 208 nm and 222
nm). The spectra of JL7short1, JL7short2 and JL7short3+4 are
indicative for proteins with a high degree of unordered structure
(signal band near 200 nm).
DETAILED DESCRIPTION OF THE INVENTION
The properties of T. cruzi JL7 antigen and its amino acid sequence
have been widely described in the state of the art. However, the
problem of increased susceptibility to interference of diagnostic
T. cruzi assays that is caused by antigens with multiple repeats
has not been addressed sufficiently. The high vulnerability to
interference can lead to false positive results when for example a
non-specific IgM molecule or a non-specific IgG together with a
rheumatic factor present in a sample to be analyzed produces a
positive test result, pretending that anti-T. cruzi antibodies are
present in a sample whereas in reality the sample should be
detected as negative. False-positive results lead to an undesired
decrease of assay specificity. However, in diagnostic methods for
infectious diseases parameters regulatory authorities request a
high degree of specificity.
Surprisingly, we have identified JL7 variants that on the one hand
do still possess sufficient antigenic properties to be bound by
sample antibodies and on the other hand do not lead to
false-positive results. The complete JL7 polypeptide harbors about
3.5 repeats of a motif of 68 amino acids. The question was whether
one repeat would be sufficient to provide a suitable diagnostic
rare reagent. When repeats 1 and 2 (designated as JL7short1, SEQ ID
NO. 2 and JL7short2, SEQ ID NO. 3, respectively) were expressed as
separate antigens none of the shortened variants was able to detect
all of the positive samples correctly (Table 2). The same was true
for a shortened variant consisting of about 1.5 repeats, called
JL7short3+4 (shown in SEQ ID NO. 5). However, when two repeats of
said 68 amino acid motif were put together adjacently such as in
SEQ ID NO. 4 samples that had been initially diagnosed as positive
using a complete JL7 polypeptide harboring 3.5 repeats turned out
to be in fact false positive samples. On the other hand, said JL7
polypeptide having two repeats of a 68 amino acid repeat as shown
in SEQ ID NO. 2 were able to reliably detect real positive samples
correctly as positive, i.e. as to contain antibodies against T.
cruzi.
The invention therefore concerns a polypeptide suitable for
detecting antibodies against Trypanosoma cruzi JL7 antigen in an
isolated biological sample comprising a JL7 specific amino acid
sequence, said JL7 specific sequence consisting of two copies of
SEQ ID NO. 2, wherein each of said two copies has an amino acid
identity of at least 90% to SEQ ID NO. 2 and wherein no further
Trypanosoma cruzi specific amino acid sequences are present in said
polypeptide. According to the invention the two copies of SEQ ID
NO. 2 can be directly adjacent to each other so that no additional
amino acid is present between the two copies. As an alternative
they can be separated by a linker sequence that is not present in
the JL7 specific sequence.
These two copies of SEQ ID NO. 2 do not need to be completely
identical but both sequences have to show an amino acid identity of
at least 90% compared to SEQ ID NO. 2. For example, SEQ ID NO. 4
(JL7short1+2) comprises SEQ ID NO. 2 (JL7short1) and SEQ ID NO. 3
(JL7short2). SEQ ID NO. 2 simply shows 100% amino acid identity to
SEQ NO. 2 and SEQ ID NO. 3 has got 66 out of 68 amino acids
identical to SEQ ID NO. 2, resulting in a degree of amino acid
identity of 97%. As another example, repeat 3 shown in FIG. 1 (SEQ
ID NO. 6) has differences in six amino acid positions compared to
SEQ ID NO. 2, resulting in 91% amino acid identity (62/68).
According to the invention the JL7 polypeptide can therefore
comprise an amino acid sequence wherein the JL7 specific portion
consists of SEQ ID NOs. 2 and 3 or SEQ ID NO. 2 and 6 or SEQ ID NOs
3 and 6. In a preferred mode the JL7 specific portion consists of
SEQ ID NO. 4 which is a combination of SEQ ID NOs 2 and 3.
For the sake of clarity, also a JL7 specific amino acid sequence of
a shorter length of 62, 63, 64, 65, 66 or 67 amino acids, wherein
all remaining amino acid residues of the shortened JL7 fragment are
identical to SEQ ID NO. 2, fulfills the requirement of at least 90%
amino acid identity to SEQ ID NO. 2, e.g. 62/68=91%. In another
embodiment the requirement of amino acid identity to SEQ ID NO. 2
is at least 95%. Preferably, the deletion of said shortened JL7
fragment concerns N- and C-terminal amino acid residues, leaving
the core of the JL7 SEQ ID NO. 2 sequence intact.
It is important that apart from the JL7 specific amino acid
sequence no further Trypanosoma cruzi specific amino acid sequences
are present in the polypeptides according to the invention. For the
sake of clarity, also further JL7 specific amino acid sequences
beyond those explicitly described are absent in the polypeptides.
Full-length JL7 as disclosed in SEQ ID NO. 1 is therefore not
encompassed in the invention. To provide clarity of the invention,
also several molecules of e.g. SEQ ID NO. 4 which already harbors
two repeats of the sequence motif shown in SEQ ID NO. 2 cannot be
present on the same polypeptide chain.
According to the invention also variants of the JL7 antigen are
contemplated as long as the prerequisite that the JL7 specific
sequence consists of two copies of SEQ ID NO.2, each having an
amino acid identity of at least 90% to SEQ ID NO. 2, is fulfilled.
In an embodiment said amino acid identity is at least 95% to SEQ ID
NO. 2. A variant classifies as such as long as the immunoreactivity
in an in vitro diagnostic immunoassay is maintained, i.e. the
variant is still able to bind and detect anti-T. cruzi antibodies
present in a sample. A variant is also a JL7 polypeptide which has
been modified for example by covalent attachment of a linker amino
acid sequence, a label, a tag, an amino acid sequence or carrier
moiety to the polypeptide or antigen. The term "variant" also
relates to a post-translationally modified protein such as a
glycosylated or phosphorylated protein or to fusion proteins that
facilitate cloning, expression, purification and folding.
The terms JL7 variant, JL7 antigen, JL7 polypeptide or JL7
antigenic polypeptide or protein are used synonymously in this
specification. Also the terms Trypanosoma cruzi (=T. cruzi)
specific antigen or T. cruzi polypeptide are understood as synonyms
and each refer to a polypeptide sequence, also referred to as amino
acid sequence, that can be found in any naturally occurring T.
cruzi strain accessible through an international protein sequence
database such as UniProt.
Usually in order to detect all stages of a T. cruzi infection
several different T. cruzi antigens are applied in an assay for
detecting T. cruzi antibodies. A further aspect of the invention is
therefore a composition of polypeptides suitable for detecting
antibodies against Trypanosoma cruzi antigens in an isolated
biological sample comprising a JL7 polypeptide as specified above
and at least one Trypanosoma cruzi polypeptide selected from the
group consisting of 1F8, Cruzipain, KMP-11 and PAR-2. Amino acid
sequences from these additional antigens are known in prior art and
are retrievable from publically available databases such as
UniProt; e.g. 1F8 (UniProt entry Q4D1Q2), also known as FCaBP, Tc24
or Tc28; Cruzipain (UniProt entry Q9TW51), also known as Cruzain,
gp51/57, Ag 163B6; KMP-11 (UniProt entry Q9U6Z1); PAR2 (UniProt
entry Q01530), also known as PFR2.
The term composition means that isolated separate T. cruzi
polypeptides are combined to an admixture. This term shall not
include polypeptides that have been recombinantly expressed or
synthesized on one single chain of amino acids so that all
polypeptides are located on just one polypeptide chain as a
multi-antigen-fusion polypeptide. In other words, multi-epitope
fusion antigens of several epitopes that naturally do not appear on
a single polypeptide chain are excluded. Rather, each of the
additional T. cruzi polypeptides 1F8, Cruzipain, KMP-11 and PAR2
are expressed on or chemically synthesized as separate polypeptide
chains. The composition is created by mixing the individual T.
cruzi polypeptides in one vessel or tube resulting in a
composition.
The composition can be liquid, i.e. the T. cruzi polypeptides are
added to a mixture in a water or buffer soluble form. Suitable
buffer ingredients are known to the person skilled in the art. Said
composition may also be solid, i.e. it comprises the T. cruzi
antigens in a lyophilized or otherwise dried form.
Moreover the current invention concerns a method of producing a
soluble and immunoreactive Trypanosoma cruzi JL7 polypeptide as
described further above, said method comprising the steps of
a) culturing host cells transformed with an expression vector
comprising operably linked a recombinant DNA molecule encoding a
Trypanosoma cruzi JL7 polypeptide,
b) expression of said Trypanosoma cruzi JL7 polypeptide and
c) purification of said Trypanosoma cruzi JL7 polypeptide.
Another aspect of the invention is a method for detecting
antibodies specific for Trypanosoma cruzi in an isolated sample
wherein a Trypanosoma cruzi JL7 polypeptide as disclosed above or a
Trypanosoma cruzi JL7 polypeptide obtained by a method defined
above or a composition of Trypanosoma cruzi polypeptides as just
described is used as a capture reagent and/or as a binding partner
for said Trypanosoma cruzi antibodies.
Yet another embodiment is method for detecting antibodies specific
for Trypanosoma cruzi in an isolated sample said method
comprising
a) forming an immunoreaction admixture by admixing a body fluid
sample with a Trypanosoma cruzi JL7 polypeptide according to the
invention or a composition already described or a Trypanosoma cruzi
JL7 polypeptide obtained by the method illustrated in a preceding
paragraph of this specification
b) maintaining said immunoreaction admixture for a time period
sufficient for allowing antibodies present in the body fluid sample
against said composition of polypeptides sample to immunoreact with
said composition of Trypanosoma cruzi polypeptides to form an
immunoreaction product; and
c) detecting the presence and/or the concentration of any of said
immunoreaction product.
In a further aspect the immunoassay methods according to the
invention are suitable for detecting T. cruzi antibodies of all
soluble immunoglobulin subclasses, including IgG and IgM as the
most relevant subclasses for Chagas diagnostics.
The invention further concerns a method for detecting antibodies
specific for Trypanosoma cruzi in an isolated sample in a so-called
double antigen sandwich format DAGS. Said method for detecting
antibodies specific for Trypanosoma cruzi in an isolated sample is
preferably carried out in a double antigen sandwich (DAGS) format.
In such an assay the ability of an antibody to bind at least two
different molecules of a given antigen with its two (IgG, IgA, IgE)
or ten (IgM) paratopes is required and utilized. In said DAGS
immunoassay the basic structures of the "solid phase antigen" and
the "detection antigen" are essentially the same so that the sample
antibody forms a bridge between two specific antigens. Both
antigens therefore have to be either identical or immunologically
cross-reactive so that one antibody is able to bind to both
antigens. The essential requirement for performing such assays is
that the relevant epitope or the relevant epitopes are present on
both antigens. One of the two antigens can be bound to a solid
phase and the other antigen carries a detectable label.
This method comprises the following steps:
a) adding to an isolated sample sample a first Trypanosoma cruzi
JL7 polypeptide which can be bound directly or indirectly to a
solid phase and said first Trypanosoma cruzi polypeptide carries an
effector group which is part of a bioaffine binding pair, and a
second Trypanosoma cruzi JL7 polypeptide and said second
Trypanosoma cruzi JL7 polypeptide carries a detectable label,
wherein said first and second Trypanosoma cruzi JL7 polypeptides
bind specifically to said anti-Trypanosoma cruzi antibodies,
b) forming an immunoreaction admixture comprising said first
Trypanosoma cruzi JL7 polypeptide, said sample antibody and said
second Trypanosoma cruzi JL7 polypeptide wherein a solid phase
carrying a corresponding effector group of said bioaffine binding
pair is added before, during or after forming the immunoreaction
admixture,
c) maintaining said immunoreaction admixture for a time period
sufficient for allowing Trypanosoma cruzi antibodies against said
first and second Trypanosoma cruzi JL7 polypeptides in the body
fluid sample to immunoreact with said first and second Trypanosoma
cruzi polypeptides to form an immunoreaction product,
d) separating the liquid phase from the solid phase
e) detecting the presence of any of said immunoreaction product in
the solid or liquid phase or both.
In a preferred mode of the described sandwich method the first
Trypanosoma cruzi JL7 polypeptide carries a biotin moiety, and the
second Trypanosoma cruzi JL7 polypeptide is labeled with an
electrochemiluminescent ruthenium complex the signal of which can
be detected.
The invention also covers the use of a Trypanosoma cruzi JL7
polypeptide or of a composition of T. cruzi specific polypeptides
or of a JL7 polypeptide obtained by a recombinant production method
defined above, all described in this specification further above,
in an in vitro diagnostic test for the detection of
anti-Trypanosoma cruzi antibodies.
Another aspect of this invention is a reagent kit for the detection
of anti-Trypanosoma cruzi antibodies, comprising a Trypanosoma
cruzi JL7 polypeptide according to the invention or a JL7
polypeptide obtained by the method of production described further
above or of a composition of a JL7 polypeptide and at least one
additional T. cruzi antigens selected from 1F8, Cruzipain, KMP-11
and PAR2.
Said kit is useful for an in vitro diagnostic test for the
detection of anti-Trypanosoma cruzi antibodies and may further
contain controls and standard solutions in separate vials as well
as additional reagents in one or more solutions or in lyophilized
form with the common additives, buffers, salts, detergents etc. and
instructions for use as known by the person skilled in the art.
The invention is further illustrated in the examples section.
EXAMPLE 1
Cloning and Purification of the Trypanosoma cruzi JL7 Antigen
Variants
In order to investigate the minimal size of the T. cruzi JL7
antigen suitable for its application in an immunodiagnostic test
several variants consisting of one or two repeat units were
generated.
Synthetic genes encoding the T. cruzi antigens as listed in table 1
were purchased from Eurofins MWG Operon (Ebersberg, Germany). On
the basis of the pET24a expression plasmid of Novagen (Madison,
Wis., USA) the following cloning steps were performed. The vector
was digested with Ndel and Xhol and a cassette comprising the
respective T. cruzi antigens were inserted. The insert of the
resulting plasmid was sequenced and found to encode the desired
protein. The amino acid sequences of the resulting proteins are
shown in the sequence protocol of the present invention. All
recombinant T. cruzi polypeptide variants contained a C-terminal
hexahistidine tag (SEQ ID NO. 7) to facilitate Ni-NTA-assisted
purification. SEQ ID NOs. are summarized in Table 1.
All T. cruzi antigens were purified according to the following
protocol. E. coli BL21 (DE3) cells harboring the expression plasmid
were grown in LB medium plus kanamycin (30 .mu.g/ml) to an
OD.sub.600 of 1, and cytosolic overexpression was induced by adding
isopropyl-.beta.-D-thiogalactosid
(IPTG) to a final concentration of 1 mM at a growth temperature of
37.degree. C. 4 hours after induction, cells were harvested by
centrifugation (20 min at 5000.times.g), frozen and stored at
-20.degree. C. For cell lysis, the frozen pellet was resuspended in
25 mM sodium phosphate pH 8.5, 6 mM MgCl.sub.2, 10 U/ml
Benzonase.RTM., 1 tablet Complete.RTM. and 1 tablet Complete.RTM.
EDTA-free per 50 ml of buffer (protease inhibitor cocktail) and the
resulting suspension was lysed by high pressure homogenization. The
crude lysate was supplemented up to 50 mM sodium phosphate, 10 mM
imidazole. After centrifugation the supernatant was applied onto a
Ni-NTA (nickel-nitrilotriacetate) column pre-equilibrated in buffer
A (50 mM sodium phosphate pH 8.5, 100 mM sodium chloride, 10 mM
imidazole). Prior to elution, the imidazole concentration was
raised to 40 mM in order to remove contaminant proteins. The
proteins were then eluted by applying an imidazole concentration of
250 mM. Finally, the proteins were subjected to size exclusion
chromatography and the protein-containing fractions was pooled and
concentrated.
TABLE-US-00008 TABLE 1 Summary T. cruzi JL7 antigen variants SEQ ID
Nos. T. cruzi antigen SEQ ID NO. JL7 1 JL7short1 2 JL7short2 3
JL7short1 + 2 4 JL7short3 + 4 5 JL7short3 6
EXAMPLE 2
Spectroscopic Measurements
Circular dichroism spectroscopy (CD) is the method of choice to
assess the secondary structure in proteins. Ellipticity in the
amide region (190-250 nm) reflects regular repetitive elements in
the protein backbone, i.e. the secondary structure.
Near-UV CD spectra were recorded with a Jasco-720
spectropolarimeter with a thermostatted cell holder and converted
to molar ellipticity. The buffer was 10 mM potassium phosphate pH
7.5. The pathlength was 0.1 cm, the protein concentration was 0.2
mg/ml. The band width was 1 nm, the scanning speed was 50 nm/min at
a resolution of 1 nm and the response was 2 s. In order to improve
the signal-to-noise ratio, spectra were measured eight timesand
averaged.
FIG. 2 shows far-UV CD spectra of JL7, JL7short1, JL7short2,
JL7short1+2 and JL7short3+4. The spectra of JL7 and JL7short1+2
point to proteins with high content of .alpha.-helical structural
elements (signal bands at 208 nm and 222 nm). The spectra of
JL7short1, JL7short2 and JL7short3+4 are indicative for proteins
with a high degree of unordered structure (signal band near 200
nm).
The finding that the well-structured variant JL7short1+2 showed the
best results in the immunoassay (example 4 and table 2) is
therefore consistent. We assume that the shorter variants
JL7short1, JL7short2 and JL7short3+4 are not able to maintain a
three-dimensional structure that maintains important recognizable
epitopes that can be bound by sample antibodies. JL7short1+2
presents relevant epitopes to sample antibodies in a better
accessible manner than the shorter variants JL7short1, JL7short2
and JL7short3+4 that do not possess two complete repeats of amino
acid sequence SEQ ID NO. 2.
EXAMPLE 3
Coupling of Biotin and Ruthenium Moieties to T. cruzi JL7 Antigen
Variants
The lysine .epsilon.-amino groups of the recombinant proteins were
modified at protein concentrations of .about.10 mg/ml with
N-hydroxy-succinimide activated biotin and ruthenium labels,
respectively. The label/protein molar ratio was adjusted to 5:1 and
15:1 for the biotin and ruthenium label conjugation, respectively.
The reaction buffer was 50 mM potassium phosphate (pH 8.5), 150 mM
KC1, 0.5 mM EDTA. The reaction was carried out at room temperature
for 30 minutes and stopped by adding buffered L-lysine to a final
concentration of 10 mM. After the coupling reaction, unreacted free
label was removed by passing the crude protein conjugate over a gel
filtration column (Superdex 200 HI Load).
EXAMPLE 4
Assessment of the Immunological Reactivity and Vulnerability to
Interference of the Recombinant T. cruzi JL7 Antigen Variants in an
Immunodiagnostic Test
The immunological reactivity of the different proteins was assessed
in an automated cobas.RTM. e601 analyzer (Roche Diagnostics GmbH).
Measurements were carried out in the double antigen sandwich
format. Thereby, the biotin-conjugate (i.e. the capture antigen) is
immobilized on the surface of a streptavidin-coated magnetic bead,
whereas the detection-antigen bears a complexed ruthenium cation as
the signaling moiety. Signal detection in cobas.RTM. e601 is based
on electrochemiluminescence.
In the presence of a specific immunoglobulin analyte, the
chromogenic ruthenium complex is bridged to the solid phase and
emits light at 620 nm after excitation at a platinum electrode. The
signal output is in arbitrary light units. Measurements were
performed with anti-T. cruzi positive and negative human serum and
plasma samples purchased from several sources.
The recombinant T. cruzi JL7 antigen variants according to the
invention were assessed pairwise in a double antigen sandwich
(DAGS) immunoassay format. For instance, a JL7-biotin conjugate was
assessed together with a JL7-ruthenium complex conjugate at a
concentration of 100 ng/ml each in assay buffer containing 50 mM
MES (pH 6.5), 150 mM NaCl, 0.1% polidocanol, 0.2% bovine albumin,
0.01% N-methylisothiazolon, 0.1% Oxy-Pyrion. The used sample volume
was 30 Anti-T. cruzi negative human sera were used as controls.
Anti-T. cruzi positive human sera were used to assess the
antigenicity of each variant.
TABLE-US-00009 TABLE 2 Detection of anti-T. cruzi antibodies in
human sera by using recombinant T. cruzi JL7 antigen variants JL7
JL7short1 JL7short2 JL7short1 + 2 JL7short3 + 4 bioelisa counts
counts counts counts counts Chagas normal samples #1 779 721 664
634 600 negative #2 714 677 549 507 519 negative #3 691 600 538 509
521 negative #4 688 592 553 513 537 negative #5 706 601 538 509 532
negative mean 716 638 568 534 542 cut-off (4.5 .times. 3'220 2'872
2'558 2'405 2'438 mean) chagas positive samples SN1440-015
8'535'719 641'038 904'786 13'345'113 30'891 positive SN1440-021
2'606'324 644 653 1'584'540 719 positive SN1440-052 3'131'565 1'429
3'487 3'807'804 744 positive SN1440-059 554'033 818 1'358 394'162
721 positive SN1440-094 2'396'405 11'031 17'289 1'801'162 650
positive reactive samples of bavarian blood donors SN1489-006
44'066 621 555 495 522 negative SN1489-201 3'618 606 564 5'811 527
negative SN1489-237 31'562 629 553 2'212 2'700 negative SN1489-553
17'161 620 548 14'993 535 negative
In Table 2, the immunological activity of all T. cruzi JL7 antigen
variants listed in Table 1 (except for JL7short3) is shown.
All samples of Table 2 were tested with a commercially available
Chagas assays (bioelisa Chagas from Biokit S. A.) that use several
T. cruzi antigens but no JL7 according to the instructions of the
manufacturer.
A specificity study comprising blood donors from the Bavarian Red
Cross (n=998) revealed 4 samples expected to be false reactive for
anti T. cruzi antibodies due to the fact that Bavaria (Germany) is
not an endemic area of the Chagas disease and also the approved
bioelisa Chagas (Biokit S. A.) was non-reactive.
In order to decide whether a sample is reactive or non-reactive a
mean background signal was determined (average of negative samples)
and a cut-off-value was calculated which was 4.5 times the mean
background signal (4.5.times.mean).
The cut-off value as threshold for determining whether a sample is
reactive or non-reactive is chosen individually depending on the
assay conditions. Such a procedure is known to a person skilled in
the art. In addition, the absolute signal counts can be normalized
by deviding the signal counts by the pre-determined cut-off value
(data not shown). Thus a positive, i.e. reactive sample would show
up as a normalized value of greater than 1 (>1) and results from
non-reactive samples would have a value between 0 and 1.
Turning to Table 2, it is obvious that the reactivity of the T.
cruzi JL7 antigen variants is strongly dependent on the number of
complete repeat units. So the isolated repeat unit 1 or 2 and the
fused repeat units 3 with the shortened unit 4 showed significant
reduced antigenicity. Its vulnerability to interference seems to be
at once strongly diminished. In contrast to this, the fusion of two
complete repeat units as in the case of the variant JL7short1+2
maintains the entire antigenicity as in the complete JL7 molecule
and at the same time identifies negative and positive samples
correctly. Two out of four interference samples showed a
significant reduction of the measured signals. Use of a JL7 variant
wherein the JL7 specific part consists of only two repeats of SEQ
ID NO. 2 (and variants with at least 90% or in an embodiment with
at least 95% amino acid sequence identity to it) increases the
specificity of an anti-T. cruzi immunoassay considerably.
SEQUENCE LISTINGS
1
71226PRTTrypanosoma cruzi 1Met Glu Gln Glu Arg Arg Gln Leu Leu Glu
Lys Asp Pro Arg Arg Asn 1 5 10 15 Ala Arg Glu Ile Ala Ala Leu Glu
Glu Ser Met Asn Ala Arg Ala Gln 20 25 30 Glu Leu Ala Arg Glu Lys
Lys Leu Ala Asp Arg Ala Phe Leu Asp Gln 35 40 45 Lys Pro Glu Gly
Val Pro Leu Arg Glu Leu Pro Leu Asp Asp Asp Ser 50 55 60 Asp Phe
Val Ala Met Glu Gln Glu Arg Arg Gln Leu Leu Glu Lys Asp 65 70 75 80
Pro Arg Arg Asn Ala Lys Glu Ile Ala Ala Leu Glu Glu Ser Met Asn 85
90 95 Ala Arg Ala Gln Glu Leu Ala Arg Glu Lys Lys Leu Ala Asp Arg
Ala 100 105 110 Phe Leu Asp Gln Lys Pro Glu Gly Val Pro Leu Arg Glu
Leu Pro Leu 115 120 125 Asp Asp Asp Ser Asp Phe Val Ser Met Glu Gln
Glu Arg Arg Gln Leu 130 135 140 Leu Glu Lys Asp Pro Arg Arg Asn Val
Gln Lys Ile Ala Asp Leu Glu 145 150 155 160 Glu Ser Met Asn Ala Arg
Ala Gln Glu Leu Ala Arg Glu Lys Lys Leu 165 170 175 Ala Asp Arg Ala
Phe Leu Asp Gln Lys Pro Glu Gly Val Ser Leu Arg 180 185 190 Glu Leu
Pro Leu Asp Asp Asp Ser Asp Phe Val Ser Met Glu Gln Glu 195 200 205
Arg Arg Gln Leu Leu Glu Lys Asp Pro Arg Lys Asn Val Gln Ile Val 210
215 220 Ala Asp 225 268PRTTrypanosoma cruzi 2Met Glu Gln Glu Arg
Arg Gln Leu Leu Glu Lys Asp Pro Arg Arg Asn 1 5 10 15 Ala Lys Glu
Ile Ala Ala Leu Glu Glu Ser Met Asn Ala Arg Ala Gln 20 25 30 Glu
Leu Ala Arg Glu Lys Lys Leu Ala Asp Arg Ala Phe Leu Asp Gln 35 40
45 Lys Pro Glu Gly Val Pro Leu Arg Glu Leu Pro Leu Asp Asp Asp Ser
50 55 60 Asp Phe Val Ala 65 368PRTTrypanosoma cruzi 3Met Glu Gln
Glu Arg Arg Gln Leu Leu Glu Lys Asp Pro Arg Arg Asn 1 5 10 15 Ala
Lys Glu Ile Ala Ala Leu Glu Glu Ser Met Asn Ala Arg Ala Gln 20 25
30 Glu Leu Ala Arg Glu Lys Lys Leu Ala Asp Arg Ala Phe Leu Asp Gln
35 40 45 Lys Pro Glu Gly Val Pro Leu Arg Glu Leu Pro Leu Asp Asp
Asp Ser 50 55 60 Asp Phe Val Ser 65 4136PRTTrypanosoma cruzi 4Met
Glu Gln Glu Arg Arg Gln Leu Leu Glu Lys Asp Pro Arg Arg Asn 1 5 10
15 Ala Arg Glu Ile Ala Ala Leu Glu Glu Ser Met Asn Ala Arg Ala Gln
20 25 30 Glu Leu Ala Arg Glu Lys Lys Leu Ala Asp Arg Ala Phe Leu
Asp Gln 35 40 45 Lys Pro Glu Gly Val Pro Leu Arg Glu Leu Pro Leu
Asp Asp Asp Ser 50 55 60 Asp Phe Val Ala Met Glu Gln Glu Arg Arg
Gln Leu Leu Glu Lys Asp 65 70 75 80 Pro Arg Arg Asn Ala Lys Glu Ile
Ala Ala Leu Glu Glu Ser Met Asn 85 90 95 Ala Arg Ala Gln Glu Leu
Ala Arg Glu Lys Lys Leu Ala Asp Arg Ala 100 105 110 Phe Leu Asp Gln
Lys Pro Glu Gly Val Pro Leu Arg Glu Leu Pro Leu 115 120 125 Asp Asp
Asp Ser Asp Phe Val Ser 130 135 590PRTTrypanosoma cruzi 5Met Glu
Gln Glu Arg Arg Gln Leu Leu Glu Lys Asp Pro Arg Arg Asn 1 5 10 15
Val Gln Lys Ile Ala Asp Leu Glu Glu Ser Met Asn Ala Arg Ala Gln 20
25 30 Glu Leu Ala Arg Glu Lys Lys Leu Ala Asp Arg Ala Phe Leu Asp
Gln 35 40 45 Lys Pro Glu Gly Val Ser Leu Arg Glu Leu Pro Leu Asp
Asp Asp Ser 50 55 60 Asp Phe Val Ser Met Glu Gln Glu Arg Arg Gln
Leu Leu Glu Lys Asp 65 70 75 80 Pro Arg Lys Asn Val Gln Ile Val Ala
Asp 85 90 668PRTTrypanosoma cruzi 6Met Glu Gln Glu Arg Arg Gln Leu
Leu Glu Lys Asp Pro Arg Arg Asn 1 5 10 15 Val Gln Lys Ile Ala Asp
Leu Glu Glu Ser Met Asn Ala Arg Ala Gln 20 25 30 Glu Leu Ala Arg
Glu Lys Lys Leu Ala Asp Arg Ala Phe Leu Asp Gln 35 40 45 Lys Pro
Glu Gly Val Ser Leu Arg Glu Leu Pro Leu Asp Asp Asp Ser 50 55 60
Asp Phe Val Ser 65 715PRTArtificial SequenceHexa-histidine tag 7Gly
Gly Gly Ser Gly Gly Gly Leu Glu His His His His His His 1 5 10
15
* * * * *